The present invention relates to a process for the production of the fine chemical in a microorgansm, a plant cell, a plant, a plant tissue or in one or more parts thereof. The invention furthermore relates to nucleic acid molecules, polypeptides, nucleic acid constructs, vectors, antisense molecules, antibodies, host cells, plant tissue, propagtion material, harvested material, plants, microorganisms as well as agricultural compositions and to their use.
Certain products and by-products of naturally-occurring metabolic processes in cells have utility in a wide array of industries, including, but not limited to, the food, feed, cosmetics, and pharmaceutical industries and agriculture. These molecules, collectively termed ‘fine chemicals’ include molecules such as vitamins for example vitamin A, D, E, K, B1, B2, B6, B12, C, pantothenic acid, biotin or folic acid; substances with vitamin-like character for example vitamin F, lipoic acid, ubiquinones, choline, myoinsositiol, vitamin U (S-methylmethionine), flavours for example vanillin, coumarin, isoeugenol, eugenol, (R)-carvone, (S)-carvone, menthol, jasmone or farnesol; nutraceuticals for example phytosterols, flavonoids, anthocyanidins, isoflavons or isoprenoids; detergents; fatty acids such as saturated fatty acids, mono unsaturated fatty acids (singular MUFA, plural MUFAS), poly unsaturated fatty acids (singular PUFA, plural PUFAS), waxes or lipids containing said fatty acids; carbohydrates for example cellulose, starch, dextrin, pectin, xanthangum, carrageenan or alginate; sugars for example monosaccharides such as glucose, fructose, manose, sorbose, ribose, ribulose, xylose, xylulose or galactose, disaccharides such as lactose, sucrose, saccharose, maltose, isomaltose or cellobiose, trisaccharides such as raffinose or maltotriose; carboxylic acids for example citric acid, α-ketoglutaric acid, ferulic acid, sinapic acid or lactic acid; carotinoids for example α-carotene, β-carotene, zeaxanthine, lutein, astaxanthine, lycopene, phyotoene or phytofluene, amino acids for example lysine, threonine, methionine, tryptophane, phenylalanine or tyrosine, cofactors for example heme or quinines, enzymes for example lipases, esterases, proteases, amylases, glucosidases etc. and other compounds [as described e.g. in Kuninaka, A. (1996) Nucleotides and related compounds, p. 561-612, in Biotechnology vol. 6, Rehm et al., eds. VCH: Weinheim, in Industial Microbiology and Biotechnology, Demain et al., second edition, ASM Press Washington, D.C. 1999, in Ullmann's Encyclopedia of Industrial Chemistry, vol. A27, Vitamins, p. 443-613 (1996) VCH: Weinheim and Ong, A. S., Niki, E. & Packer, L. (1995) Nutrition, Lipids, Health, and Disease Proceedings of the UNESCO/Confederation of Scientific and Technological Associations in Malaysia, and the Society for Free Radical Research, Asia, held Sep. 1-3, 1994 at Penang, Malaysia, AOCS Press, (1995)), enzymes, and all other chemicals described in Gutcho (1983) Chemicals by Fermentation, Noyes Data Corporation, ISBN: 0818805086 and references contained therein]. Carotinoids are added for example to soft drinks, margarines or to animal feed for example to colour egg yolk or the flesh of fish. In the food industry polycarbohydrates are widely used as thickener. Polyunsaturated fatty acids are added for example to infant formulas to create a higher nutrition value of such formulas. PUFAs have for example a positive influence on the cholesterol level of the blood in humans and therefore are useful in the protection of heart diseases. Fine chemicals for example PUFAS can be isolated from animal sources such as for example fish or produced with microorganisms through the large-scale culture of microorganisms developed to produce and accumulate or secrete large quantities of one or more desired molecules.
In large scale fine chemicals are produced with microorganism in the fermentation industry, which is responsible for the manufacturing of at least five major ingredient categories: antibiotics, organic acids, amino acids, enzymes, vitamins and other related products. There are production facilities in all important areas of the world especially in Europe, the US and Asia. Companies continuously try to optimize the production processes, the organisms and thereby increasing the efficiency but, as in the case of amino acids and organic acids, with already high conversion rates based on feeded carbon source, the limitations of such work become evident. All fermentation processes depend on the efficient utilization of carbohydrates, supplied mainly in the form of oils, glucose or molasses. It is therefore the availability and pricing of these raw materials that influence the competitiveness of fermentation products versus production for example in plants. Amino acids, organic acids and vitamins are offered at very low prices. For such products the question is whether it is still economical to continue fermentation production in future. And that is frequently a question of comparing the availability and pricing of carbohydrates with the future markets.
Particularly useful organisms for the production of fine chemicals are microorganisms such as the algae, fungi, bacteria or plants. Through strain selection, a number of mutant strains of microorganisms have been developed which produce an array of desirable compounds including vitamins, amino acids, PUFAs etc. However, selection of strains improved for the production of a particular molecule is a time-consuming and difficult process.
Alternatively the production of fine chemicals can be most conveniently performed via the large scale production of plants developed to produce for example carotinoids, carbohydrates or PUFAS. For example for the production of carotinoids plants such as marigold are used. Particularly well-suited plants for this purpose are sugar producing plants such as sugar beet or sugar cane or oilseed plants containing high amounts of lipid compounds such as rapeseed, canola, linseed, soybean, sunflower, borage and evening primrose. But also other crop plants containing sugars, oils or lipids and fatty acids are well suited as mentioned in the detailed description of this invention. Through conventional breeding, a number of mutant plants have been developed which produce an array of desirable lipids and fatty acids, cofactors and enzymes. However, selection of new plant cultivars improved for the production of a particular molecule is a time-consuming and difficult process or even impossible if the compound does not naturally occur in the respective plant as for example in the case of C20 and higher C-carbon chain polyunsaturated fatty acids.
Carbohydrates are an important dietary nutrient, which is mostly used to supply energy to the body, as well as, a carbon source for synthesis of other compounds such as fats or proteins. Furthermore mono- and disaccharides are widely used in the food and feed industry as sweetener. Saccharides have varying degrees of sweetness on a relative scale. Fructose is the sweetest. For example in the United States 22 million tons of sugar and other sweeteners were consumed in 1999. Said natural sweetener consumption includes the consumption of sugar, corn sweeteners such as high fructose corn syrup as main product and others such as honey or maple syrup. All natural sugar based sweeteners have a market shar of around 36 to 40 percent.
Whether unsaturated or saturated fatty acids are preferred in the food and feed industry depends on the intended purpose; thus, for example, lipids with unsaturated fatty acids, specifically polyunsaturated fatty acids, are preferred in human nutrition since they have a positive effect on the cholesterol level in the blood and thus on the possibility of heart disease. They are used in a variety of dietetic foodstuffs or medicaments. In addition PUFAs are commonly used in food, feed and in the cosmetic industry. Poly unsaturated ω-3- and/or ω-6-fatty acids are an important part of animal feed and human food. Because of the common composition of human food poly unsaturated ω-3-fatty acids, which are an essential component of fish oil, should be added to the food to increase the nutritional value of the food; thus, for example, poly unsaturated fatty acids such as docosahexaenoic acid or eicosapentaenoic acid are added as mentioned above to infant formula to increase its nutritional value.
Vitamins such as vitamin C, vitamin B12 or vitamin B2 are typically produced with microorganism as mentioned above in the fermentation industry. Vitamin C can be produced generally in a combined process using biotransformation steps in combination with classical chemical synthesis. In another production process vitamin C is produced by fermentation alone. In general organisms such as Arthrobacter, Gluconobacter, Corynebacterium, Brevibacterium or Erwinia are used for vitamin C production. Vitamin B2 and vitamin B12 are produced with organisms such as Bacillus, Streptomyces, Citrobacter, Klebsiella, Propionibacterium or Ashbya in large scale fermentation.
Commonly vitamin E and A are procuded in a classical chemical process or isolated from as natural vitamin E from plant oils. Vitamin E is an important natural fat-soluble antioxidants. As such, vitamin E protects cell membranes from the damage caused by free radicals. High doses of vitamin E have also been linked to a decreased ability of the blood to clot, which may be beneficial in those individuals at risk for heart disease by reducing the risk of heart attack. A vitamin E deficiency leads to pathophysiological situations in humans and animals. Of the different types of vitamin E, the alpha tocopherol form is typically considered the “gold standard” in terms of antioxidant activity—although the most recent research suggests that the other chemical forms may possess equivalent or superior antioxidant protection. Vitamin E compounds therefore are of high economical value as additives in the food and feed sectors, in pharmaceutical formulations and in cosmetic applications. Vitamin A is another fat-soluble vitamin that is part of a family of compounds including retinol, retinal and beta-carotene. Beta-carotene is also known as pro-vitamin A because it can be converted into vitamin A when additional levels are required. Vitamin A is needed by all of the body's tissues for general growth and repair processes and is especially important for bone formation, healthy skin/hair, night vision and function of the immune system. Vitamin A may help boost immune system function and resistance to infection. Vitamin A derivatives are widely used in cosmetics and dermatological treatments for skin preparations designed to combat skin aging and treat acne. Vitamin A has been used for decades as a treatment for various vision-related conditions, including night blindness, cataracts, conjunctivitis, retinopathy and macular degeneration.
An economical method for producing of vitamins such as vitamin C, B2, B12 or vitamin E and food- and feedstuffs with increased vitamin content are therefore very important. Particularly economical methods are biotechnological methods utilizing vitamin-producing organisms, which are either natural or optimized by genetic modification.
Carotenoids are a large family of compounds including over 600 members such as β-carotene, lycopene or lutein. Carotenoids are widely distributed in fruits and vegetables and are responsible, along with flavonoids, for contributing the color to many plants (a rule of thumb is the brighter, the better). In terms of nutrition, β-carotene's primary role is as mentioned above a precursor to vitamin A. β-carotene as most other carotenoids, is a powerful antioxidant—so it has been recommended to protect against a variety of diseases such as cancer, cataracts and heart disease.
The introduction of a new gene or new genes for the synthesis of fine chemicals into an organism or cell may not just increase the biosynthetic flux towards an end product it may also increase or create de novo a new compound composition. Similarly, other genes involved in the import of nutrients necessary for the biosynthesis of one or more fine chemicals (e.g., fatty acids, polar and neutral lipids, vitamins, enzymes etc.) may be increased in number or activity such that these precursors, cofactors, or intermediate compounds are increased in concentration within the cell or within the storing compartment thus increasing further the capability of the cell to produce the fine chemical as described herein.
Amino acids are used in many branches of industry, including the food, animal feed, cosmetics, pharmaceutical and chemical industries. Amino acids such as D,L-methionine, L-lysine or L-threonine are used in the animal feed industry. The essential amino acids valine, leucine, isoleucine, lysine, threonine, methionine, tyrosine, phenylalanine and tryptophan are particularly important for the nutrition of mammals especially humans and a number of livestock species. Glycine, L-methionine and tryptophan are all used in the pharmaceutical industry. Glutamine, valine, leucine, isoleucine, histidine, arginine, proline, serine and alanine are used in the pharmaceutical and cosmetics industries. Threonine, tryptophan and D,L-methionine are widely used feed additives (Leuchtenberger, W. (1996) Amino acids—technical production and use, pp. 466-502 in Rehm et al., (Ed.) Biotechnology vol. 6, chapter 14a, VCH Weinheim). Moreover, amino acids are suitable for the chemical industry as precursors for the synthesis of synthetic amino acids and proteins, such as N-acetylcysteine, S-carboxymethyl-L-cysteine, (S)-5-hydroxytryptophan and other subtances described in Ullmann's Encyclopedia of Industrial Chemistry, vol. A2, pp. 57-97, VCH Weinheim, 1985. To prefent physiological malnutritions the human body has a need for essentiall amino acids such as arginine, histidine, isoleucine, leucine, lysine, methionine, tyrosine, phenylalanine, threonine, tryptophan, and valine. Based on their content of amino acids, foods are often classified as complete, partially complete, or incomplete protein sources. In order for a protein to be complete, it must contain all of the essential amino acids. This is the reason that many nutritionists rank non-meat foods as being incomplete. The foods do contain all amino acids, but some may be in lower proportions than are required, and, therefore, should be combined with another food containing higher amounts of these amino acids or should be supplemented with said essential amino acids.
Over one million tonnes of amino acids are currently produced annually; their market value amounts to over 2.5 billion US dollars. They are currently produced by four competing processes: Extraction from protein hydrolysates, for example L-cystine, L-leucine or L-tyrosine, chemical synthesis, for example of D,L-methionine, conversion of chemical precursors in an enzyme or cell reactor, for example L-phenylalanine, and fermentative production by growing, on an industrial scale, bacteria which have been developed to produce and secrete large amounts of the desired molecule in question. An organism, which is particularly suitable for this purpose is Corynebacterium glutamicum, which is used for example for the production of L-lysine or L-glutamic acid. Other amino acids which are produced by fermentation are, for example, L-threonine, L-tryptophan, L-aspartic acid and L-phenylalanine.
The biosynthesis of the natural amino acids in organisms capable of producing them, for example bacteria, has been characterizied thoroughly; for a review of the bacterial amino acid biosynthesis and its regulation, see Umbarger, H. E. (1978) Ann. Rev. Biochem. 47: 533-606].
It is known that amino acids are produced by fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum. Due to their great importance, the production processes are constantly being improved. Process improvements can relate to measures regarding technical aspects of the fermentation, such as, for example, stirring and oxygen supply, or the nutrient media composition, such as, for example, the sugar concentration during fermentation, or to the work-up to give the product, for example by ion exchange chromatography, or to the intrinsic performance properties of the microorganism itself. Bacteria from other genera such as Escherichia or Bacillus are also used for the production of amino acids. A number of mutant strains, which produce an assortment of desirable compounds from the group of the sulfur-containing fine chemicals have been developed via strain selection. The performance properties of said microorganisms are improved with respect to the production of a particular molecule by applying methods of mutagenesis, selection and mutant selection. Methods for the production of methionine have also been developed. In this manner, strains are obtained which are, for example, resistant to antimetabolites, such as, for example, the methionine analogues α-methylmethionine, ethionine, norleucine, N-acetylnorleucine, S-trifluoromethylhomocysteine, 2-amino-5-heprenoitic acid, selenomethionine, methionine sulfoximine, methoxine, 1-aminocyclopentanecarboxylic acid or which are auxotrophic for metabolites with regulatory importance and which produce sulfur-containing fine chemicals such as, for example, L-methionine. However, such processes developed for the production of methionine have the disadvantage that their yields are too low for being economically exploitable and that they are therefore not yet competitive with regard to chemical synthesis.
Zeh (Plant Physiol., Vol. 127, 2001: 792-802) describes increasing the methionine content in potato plants by inhibiting threonine synthase by what is known as antisense technology. This leads to a reduced threonine synthase activity without the threonine content in the plant being reduced. This technology is highly complex; the enzymatic activity must be inhibited in a very differentiated manner since otherwise auxotrophism for the amino acid occurs and the plant will no longer grow.
U.S. Pat. No. 5,589,616 teaches the production of higher amounts of amino acids in plants by overexpressing a monocot storage protein in dicots. WO 96/38574, WO 97/07665, WO 97/28247, U.S. Pat. No. 4,886,878, U.S. Pat. No. 5,082,993 and U.S. Pat. No. 5,670,635 are following this approach. That means in all the aforementioned intellectual property rights different proteins or polypeptides are expressed in plants. Said proteins or polypeptides should function as amino acid sinks. Other methods for increasing amino acids such as lysine are disclosed in WO 95/15392, WO 96/38574, WO 89/11789 or WO 93/19190. In this cases speziell enzymes in the amino acid biosynthetic pathway such as the diphydrodipicolinic acid synthase are deregulated. This leads to an increase in the production of lysine in the different plants. Another approach to increase the level of amino acids in plants is disclosed in EP-A-0 271 408. EP-A-0 271 408 teaches the mutagenensis of plant and selection afterwards with inhibitors of certain enzymes of amino acid biosynthetic pathway.
Methods of recombinant DNA technology have also been used for some years to improve Corynebacterium strains producing L-amino acids by amplifying individual amino acid biosynthesis genes and investigating the effect on amino acid production.
As described above, the essential amino acids are necessary for humans and many mammals, for example for livestock. L-methionine is important as methyl group donor for the biosynthesis of, for example, choline, creatine, adrenaline, bases and RNA and DNA, histidine, and for the transmethylation following the formation of S-adenosylmethionine or as a sulfhydryl group donor for the formation of cysteine. Moreover, L-methionine appears to have a positive effect in depression.
Improving the quality of foodstuffs and animal feeds is an important task of the food-and-feed industry. This is necessary since, for example, certain amino acids, which occur in plants are limited with regard to the supply of mammals. Especially advantageous for the quality of foodstuffs and animal feeds is as balanced as possible an amino acid profile since a great excess of an amino acid above a specific concentration in the food has no further positive effect on the utilization of the food since other amino acids suddenly become limiting. A further increase in quality is only possible via addition of further amino acids, which are limiting under these conditions. The targeted addition of the limiting amino acid in the form of synthetic products must be carried out with extreme caution in order to avoid amino acid imbalance. For example, the addition of an essential amino acid stimulates protein digestion, which may cause deficiency situations for the second or third limiting amino acid, in particular. In feeding experiments, for example casein feeding experiments, the additional provision of methionine, which is limiting in casein, has revealed the fatty degeneration of liver, which could only be alleviated after the additional provision of tryptophan.
To ensure a high quality of foods and animal feeds, it is therefore necessary to add fine chemicals that means a plurality of compounds such as amino acids, vitamins, organic acids, PUFAS etc. in a balanced manner to suit the organism. Such supplemented food is named as “functional foods” or “nutraceuticals”. Nutraceuticals shall provide a health benefit to humans beyond basic nutrition. Functional foods have health-promoting or disease-preventing effects. Examples include omega-3 fatty acids (found in many fish, flaxseed oil, soybean oil, canola oil, and walnuts), which reduce risk of coronary heart disease and lycopene in tomatoes, which has been associated with reduced risk of certain cancers.
From a practical standpoint it would be of great advantage to produce an organism such as a microorganism or a plant containing a combination of different fine chemicals such as amino acids, vitamins, organic acids, carotenoids, PUFAS etc. at the same time in an sufficient amount to provide optimal growth and health benefit to animals or humans instead of combining different food or supplementing food or feed with different fine chemicals.